Date of this version: Jun 22 2021

Project goal

A reviewer of a project of ours on colour learning says the following:

The critical problem of this study is the presence of covariation between (at least) three features of the object stimulus, its colour, its size, and its shape. Therefore, it is not possible to conclude whether the observed difference in the learned preference between the two stimuli was due to the colour or to the shape or something else.

In order to address this point we are re-doing the study with this critique in mind. We are conducting a study where the goal is to determine whether guppies can change their preference for a coloured object, regardless of its colour. Guppies are trained to green and blue objects that differ only in their colours and not also their sizes and shapes (Figure 1).

Protocol overview

In this overview steps 1 - 3 are largely similar to experiment 1. This time the experiment is done with a large variety of objects that only differ in their colour. Steps 4 - 6 are additional trials for this second experiment so that, in addition to making sure learning is specific to colour, we also see how robust learning to colour is across different objects of similar colours.

  1. Initial test: 1 unreinforced preference trial with object pair A. An empty food strip is attached to each object.
  2. Training: 20 reinforced trials with object pairs 1-4. A rewarded food strip is attached to the rewarded object, and an empty food strip to the other object.
  3. Final test: 1 post-training, unreinforced preference trial with object pair A. An empty food strip is attached to each object.
  4. Refresher trials: 2 reinforced trials with object pairs 2 and 3
  5. Generalization 1: 1 unreinforced preference trial with object pair B. An empty food strip is attached to each object.
  6. Generalization 2: 1 unreinforced preference trial with object pair C. An empty food strip is attached to each object.

During training, on any given day all guppies will get reinforced for visiting the same object pair. The presentation of the four training object pairs will have a randomized order that is the same for all guppies.

Objects are cleaned by rinsing with water conditioned with SeaChem Prime between trials. For example, if a single training object was used, a scratch could mean that particular object was treated differently from other objects, so we would be looking at learning about a scratch, not colour.

Generalization 1 refers to a generalization to different colours of Lego material whereas Generalization 2 refers to generalization to completely different objects (green and blue disks).

The 7 pairs of objects for the experiment as well as an experimental timeline. The numbered object pairs are used for training while the lettered object pairs are used for testing. The experimental timeline represents the order of the trials. The open orange square at trial 0 represents the initial test which is done with object pair A. The filled green circles represent the 20 training trials (trials 1-20). The filled orange square is the final test trial which is done with object pair A. The 2 red circles represent the refresher trials which are done with object pairs 2 and 3 (I picked these with a random number generator between 1 and 4). The open cyan triangle represents generalization 1 which is done with object pair B. The open blue circle is generalization 2 which is done with object pair C

Figure 1: The 7 pairs of objects for the experiment as well as an experimental timeline. The numbered object pairs are used for training while the lettered object pairs are used for testing. The experimental timeline represents the order of the trials. The open orange square at trial 0 represents the initial test which is done with object pair A. The filled green circles represent the 20 training trials (trials 1-20). The filled orange square is the final test trial which is done with object pair A. The 2 red circles represent the refresher trials which are done with object pairs 2 and 3 (I picked these with a random number generator between 1 and 4). The open cyan triangle represents generalization 1 which is done with object pair B. The open blue circle is generalization 2 which is done with object pair C

Materials information

Colours

Object Colour Colour Family Exact Colour Hex Value
Green Dark Green Bright Yellowish Green B5BD00
Blue Blue Dark Azur

Bricks

The objects were 6 pairs of Lego blocks with the manufacturer colours A and B. The specific models for the object pairs are as follows

Object Pair Brick Style Design ID
Pair A BRICK 1X2 3004
Pair B BRICK
Pair 1 BRICK 1X2 3004
Pair 2 BRICK 2X4 3001
Pair 3 BRICK 2X2 30165
Pair 4 BRICK 1X1 3005

These can be looked up and/or purchased using the official Lego store’s Choose a brick service by entering the design ID in the Enter an item number or design ID prompt of the Enter set number section.

Detailed protocol

In this section we provide step by step instructions on how to perform an experiment for the day.

Overview

Briefly the protocol involves the following: First you need to make the food strips and then wait one hour for them to fix onto the strips. Then you run the trials with the fish. Each trial lasts 5 minutes. Guppies are tested and trained in the same order every day.

Throughout all trials the side of the tank the rewarding object is on should be switched. I have written an R script that has produced a data sheet which randomly assigned individuals to treatments as well as which sides of the tank should be rewarded during training for each guppy.

The script ensures on any given day the position of the object is random but across all trials the number of left reinforced and right reinforced trials is even across guppies on any given day as well within an individual guppy across all days. Consult this sheet when performing the experiment to know which objects go where for which fish on any particular day.

After performing the trials for the day you transfer the video files from the SD card on the camera and into the C://Users/Reader Lab/2-colour-learning-experiment-2/videos/ folder on the desktop. Finally, you use EthoVision to extract the positional data from the video files.

Some additional details. Guppies should only be fed Tetramin flake food in their home tank every three days during this experiment. This is to increase the motivation to learn and perform the task.

Step 1 - Food strips

The first thing to do during a training day is to make food strips. 16 strips are prepared, one for each guppy. The food strips that are attached to the objects are made with the following protocol:

  1. Pour 50 mL of conditioned water into a 100 mL beaker.
  2. Add half a serving of decapsulated brine shrimp egg to the water and stir then add a pinch of Tetramin flake food and stir.
  3. Add a packet of Knox gelatin to the water and stir for about 15 seconds or until the powder fully dissolves, then wait another 15 seconds.
  4. Use a transfer pipette to transfer the food mixture from the beaker onto X cm by Y cm strips of plastic.
  5. Spread another pinch of flake food on top of the food mixture on the plastic strips while it is still wet.
  6. Wait one hour for food to fix onto the plastic strips

Step 2 - Preparing the SD card

Before you start performing the trials the SD card needs to have footage from the previous day cleared up.

  1. Open the folder navigation menu by clicking the folder icon on the bottom left of the screen
  2. Under Computer click Removable Disk (L:) 3, Double click MP_ROOT then 100ANV01
  3. Scroll down to the files that have a Date modified date that corresponds to the previous day
  4. Hold down CTRL while selecting the files that have a type of MP4 Video
  5. Right click and select Delete
  6. Again, under Computer click Removable Disk (L:)
  7. Go to PRIVATE/AVCHD/BDMV/STREAM/
  8. Scroll down to the files that have a Date modified date that corresponds to the previous day
  9. Hold down CTRL while selecting the files that have a type of MP4 Video
  10. Right click and select Delete
  11. Under Computer right click Removable Disk (L:) and select eject
  12. Take the SD card out of the card reader and put it into the camera

Step 3 - Performing the test or training trials

If the current trial is a training trial you do the following:

  1. Place the test tank on the designated sharpie lines on the lab bench
  2. Fill the test tank with water from several home tanks to the 6 cm line using the aquaneering rack tank.
  3. Transfer the guppy from the home tank to the test tank which has been filled with water from several guppy home tanks to a level of 6 cm via a white opaque plastic cup where the green and blue objects have already been placed into the test tank. The objects should be placed on their designated velcro piece which are each 6 cm from the side of the tank with one on the left and right end of the tank. The two objects are 38 cm apart.
  4. Dip the food strip in some water to remove excess flakes. Keep dipping until the strip does not shed flakes upon being dipped in water (about 8 to 10 dips)
  5. Attach a food strip to the back of the correct rewarding object for that trial using blunt tweezers. Consult the data sheet to confirm which guppy is rewarded to which object and which side of the tank the rewarding object should be placed.
  6. Gently pour the guppy into a 20 cm tall, open-ended glass holding cylinder (7 cm diameter). Allow the guppy to acclimate in the holding cylinder for 120 seconds.
  7. Press the red Record button on the remote for the camera
  8. After acclimation the release glass should be slowly lifted and the guppies given 5 minutes to move about the tank and feed from the objects. Set a timer for 5 minutes and 20 seconds and leave the room once you have lifted the cup. Come back after 5 minutes and 20 seconds.
  9. Stop the video recording using the remote.
  10. Transfer the guppy back to the home tank via the white opaque plastic cup.
  11. Repeat steps 3 to 8 for all guppies.
  12. Remove the objects from the test tank and empty the water into the sink.

If the trial is a test trial you do the above EXCEPT there is no need to add food to a plastic strip. Instead use never before used blank plastic strips that I provide. On test trial days you therefore do not need to wait an extra hour.

Step 4 - EthoVision

  1. Carefully remove the SD card from the camera and place it into the card reader on the wet lab computer.
  2. Navigate to the video files which are located in L://MP_ROOT/
  3. Copy the 16 .mp4 video files from the trials of the current day into a folder named after the trial number (e.g., trial-x/) found within the C://Users/Reader Lab/2-colour-learning-experiment-2/videos/ folder.
  4. Rename the .mp4 files based on the IDs of the fish. Each video file should be named after a specific fish.
  5. Open EthoVision XT 11.5
  6. Under New experiment select New from template
  7. Select Use a custom template
  8. Select Browse... and navigate to C://Users/Reader Lab/2-colour-learning-experiment-2/trial-2/trial-2.evxt
  9. Click OK
  10. Click Browse... and navigate to C://Users/Reader Lab/2-colour-learning-experiment-2/ and name the file trial-x where x is the number of the trial for that day (this can be found on the data sheet).
  11. Click the Arena Settings tab and select Arena Settings 1
  12. Go to the panel on the right and right click Background.
  13. Navigate to the first video file for that day and click Open.
  14. Click Grab and make sure the arena graphics still line up then click Validate Setup.
  15. Click Trial List
  16. Click Add Trials in the top left corner and add 16 trials
  17. Click Acquisition (0 acquired)
  18. Scroll through the video footage until the point where the release cylinder is removed from the tank
  19. Wait 20 seconds to allow the water to calm
  20. Click the small camera icon on the left of the Playback Control to set the background image.
  21. Click the red circle to start processing the video.
  22. Watch for any errors in tracking and make note of when these occur
  23. Record whether the guppy eats from the rewarded object during the trial on the data sheet
  24. Click ... next to Video under the Settings tab to the right
  25. Select the next video
  26. Repeat steps 18-25 for all videos
  27. Go to the trial list and fill in the variables
  28. Perform track error correction
  29. Under the Analysis tab go to the Results tab and click Statistics & Charts
  30. Click Calculate
  31. Under the Export tab click Statistics
  32. Click OK, the default settings should be sufficient
  33. Navigate to C://Users/Reader Lab/2-colour-learning-experiment-2/trial-x/Export Files/
  34. Open Google chrome and click the third bookmark tab which contains the Google drive folder named experiment-2-raw-data/
  35. Drag the raw data file Statistics-trial-x into the Google drive folder.
  36. Save EthoVision and close it.